Liquid pharmaceutical formulation

ABSTRACT

The present invention relates to a liquid pharmaceutical formulation, which is stable at room temperature, being essentially free of water, comprising a) at least one easily degradable active pharmaceutical ingredient, b) at least one pharmaceutically acceptable organic solvent and c) at least one pharmaceutically acceptable alkaline earth metal salt and its use in medicine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Pat. Application No.17/042,795 filed Sep. 28, 2020, which is the U.S. National Stage Pat.Application of International Application No. PCT/EP2019/057984, filedMar. 28, 2019, which claims the right of priority of Luxemburg PatentApplication No. LU100750, filed Mar. 28, 2018, the contents of eachbeing hereby incorporated by reference it its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to a liquid pharmaceutical formulation,being essentially free of water, comprising a) at least one easilydegradable active pharmaceutical ingredient b) at least onepharmaceutically acceptable organic solvent and c) at least onepharmaceutically acceptable alkaline earth metal salt and its use inmedicine.

BACKGROUND

Certain pharmaceutical ingredients are degradable e.g. due tohydrolysis. An exemplary group of degradable pharmaceutical ingredientsare alkylating agents which are used for the treatment of variouscancers. One substance of this group, for example is Bendamustine.Bendamustine comprises an alkylating —N((CH₂)₂Cl)₂ group, whichundergoes rapid hydrolysis, by substitution of the chloride groups, tothe corresponding mono- and di-hydroxy compounds. The hydrolysis ofBendamustine in water takes place in hours, therefore a solution ofBendamustine is not suitable for long term storage. Bendamustine iscommercially available as powder in lyophilized form as Treanda™. Whilethe lyophilized form exhibits good chemical stability, with implicationsof chemical stability. Thus, efforts have been made to stabilizeBendamustine in liquid pharmaceutical formulations, which may be storedfor longer time.

In this respect, WO2011/094565 discloses a formulation for long termstorage including bendamustine or a pharmaceutically acceptable saltthereof; a pharmaceutically acceptable fluid including polyethyleneglycol PEG, propylene glycol (PG) and a stabilizing amount of anantioxidant such as thioglycerol. Another formulation includesbendamustine or a pharmaceutically acceptable salt thereof; apharmaceutically acceptable fluid including polyethylene glycol PEG),propylene glycol (PG), ethanol, benzyl alcohol and glycofurol and achloride salt such as sodium chloride, choline chloride, andhydrochloride salts of amino acids.

WO 2013/112762 A1 deals with aqueous Bendamustine formulations withimproved stability. These comprise a mixture of a non-aqueous solventsystem and an aqueous chloride-containing water phase. The non-aqueoussolvent may comprise propylene glycol or polyethylene glycol andoptionally antioxidants and preservatives such as thioglycerol.

The aim of the present invention is therefore to provide an optimizedpharmaceutical formulation for stabilizing easily degradable activepharmaceutical ingredients.

SUMMARY OF THE INVENTION

The Invention relates to a liquid pharmaceutical formulation, beingessentially free of water, comprising

-   a) at least one easily degradable active pharmaceutical ingredient,-   b) at least one pharmaceutically acceptable organic solvent and-   c) at least one pharmaceutically acceptable alkaline earth metal    salt.

Further, the invention is directed to the pharmaceutical formulation foruse in medicine as well as to the pharmaceutical formulation for use inthe treatment of cancer.

It has been shown that the inventive pharmaceutical formulation issuitable to stabilize easy degradable active pharmaceutical ingredientsin comparison to samples which did not comprise at least onepharmaceutically acceptable alkaline earth metal salt. An embodimentwherein the alkaline earth metal salt is CaCl₂ proved to be particularlyeffective for stabilizing easy degradable active pharmaceuticalingredients. Further, the inventive pharmaceutical formulations reduceor prevent color change indicating degradation during storage, inparticular in comparison with other pharmaceutical formulations for thesame purpose of stabilizing degradable active pharmaceuticalingredients, known in the art. Unlike organic preservatives andantioxidants the addition of alkaline earth metal salts from calcium ormagnesia in the amounts described herein are regarded as physiologicallysafe and do not require any verification. For example, in contrast tothe formulation as disclosed in WO2011/094565, the pharmaceuticalformulation of the present invention does not require the presence oftoxic thioglycerol as antioxidant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the results of the Bendamustine-HCl solubility studyaccording to example 2. Target concentration was 80 mg/g after storageover-night at 2-8° C. and subsequent centrifugation. Analysis wasperformed by RP-HPLC.

FIG. 2 depicts purity at 25° C. of Bendamustine-HCl in the formulationvariants determined by relative main peak evaluation in RP-HPLCchromatograms.

FIG. 3 depicts Purity at 40° C. of Bendamustine-HCl in the formulationvariants determined by relative main peak evaluation in RP-HPLCchromatograms.

FIG. 4 depicts content at 25° C. of Bendamustine-HCl in the formulationvariants determined by relative main peak evaluation in RP-HPLCchromatograms.

FIG. 5 depicts Purity at 40° C. of Bendamustine-HCl in the formulationvariants determined by relative main peak evaluation in RP-HPLCchromatograms.

FIG. 6 depicts Photographs of the Bendamustine-HCl stability samplestaken after the end of the stability study. Top: stored at 25° C.;Bottom: stored at 40° C.

FIG. 7 depicts the Bendamustine purity by RP-HPLC

FIG. 8 depicts the Rel. Bendamustine concentration (ref. T = 0) byRP-HPLC

FIG. 9 depicts the Azacitidine purity by RP-HPLC

FIG. 10 depicts rel. Azacitidine concentration (ref. T = 0) by RP-HPLC

FIG. 11 depicts Carmustine purity by RP-HPLC

FIG. 12 depicts rel. Carmustine concentration (ref. T=0) by RP-HPLC

FIG. 13 depicts Melphalan purity by RP-HPLC

FIG. 14 depicts rel. Melphalan concentration (ref. T = 0) by RP-HPLC

FIG. 15 depicts the purity of Bendamustine-HCl at 40° C. after 8 weeksstorage time shown as relative main peak area by RP-HPLC. Displayed arecalculated mean values of duplicate measurements, relative to thestarting value at T=0.

FIG. 16 depicts the content of Bendamustine-HCl at 40° C. after 8 weeksstorage time by RP-HPLC. Displayed are calculated mean values ofduplicate measurements. Target: 25 mg/g.

FIG. 17 depicts the purity of Bendamustine-HCl at 40° C. after 8 weeksstorage time by RP-HPLC

FIG. 18 depicts content of Bendamustine-HCl at 40° C. after 8 weeksstorage time by RP-HPLC

FIG. 19 : The visual appearance of the liquid formulations wasdocumented by macro photography. Photographs of representative vialsafter 8 weeks storage time at 40° C. are displayed.

DETAILED DESCRIPTION OF THE INVENTION

The solution of the present invention is described in the following,exemplified in the appended examples, illustrated in the Figures andreflected in the claims.

The present invention provides a liquid pharmaceutical formulation,being essentially free of water, comprising

-   a) at least one aromatic nitrogen mustard active pharmaceutical    ingredient,-   b) at least one pharmaceutically acceptable organic solvent and-   c) at least one pharmaceutically acceptable alkaline earth metal    salt.

In the context of the present invention, “essentially free of water”means a water content below 5% (w/w), preferably below 2% (w/w), morepreferably below 1% (w/w), most preferably below 0.5% (w/w).

The at least one easily degradable active pharmaceutically ingredientmay be any pharmaceutically active ingredient known to the personskilled in the art, which decomposes, is chemically transformed and/orloses its pharmaceutical activity and/or forms detrimental side productsdue to its degradation during storage in dissolved form. The easilydegradable active pharmaceutically ingredient may be used in form of itspharmaceutically acceptable salt, wherein the anion may be inorganic ororganic. For example in form of its hydrochloride. In the context of thepresent invention, the term “degradable active pharmaceuticallyingredient” refers to the neutral form and/or the correspondingpharmaceutically and/or pharmaceutically acceptable salt thereof. In thecontext of the invention, the term “pharmaceutically acceptable” meansthat the respective substance fulfills at least the necessary legalqualitative requirements for its administration to humans or animals aspart of a medical or non-medical treatment.

Preferably, the degradable pharmaceutically active ingredient is atleast sensitive to hydrolysis, for example caused by moisture, and/orthe degradable pharmaceutically active ingredient is at least sensitiveto oxidation, for example caused by oxygen or free radicals, morepreferably the degradable pharmaceutically active ingredient is at leastsensitive to hydrolysis.

Hydrolysis in the context of the invention means cleavage of one or morebonds by addition of at least one water or alcohol (HO—R) molecule orreplacement of a functional group by a hydroxyl group originating fromthe water molecule. The hydrolysis may include formation of smallmolecules formed of protons originating from the water or alcoholmolecule attached and the group which has been released is for exampleHCI or HBr.

Degradation of nitrogen mustards (ternary 2,2′-dichloroalkylamines) issupposed to take place via the formation of a reactive three-membercyclic intermediate towards mono and dihydroxyl products (e.g.hydrolysis or alcoholysis). To start this reaction a free election pairat the nitrogen atom has to be accessible. The presence of positivelycharged calcium (or other alkaline earth metal) ions reduces theaccessibility of the partial negatively charged election pair at thenitrogen atom by charge interactions between the calcium ion and thefree electron pair. By masking the said electron pair at the nitrogenatom with calcium ions the cyclization reaction is slowed down orcompletely blocked resulting in a demonstrable stabilizing effect onnitrogen-mustard compounds:

The stabilizing effect is depending on the capability of the alkalineearth metal ion to undergo charge interactions to the election pair atthe nitrogen atom. Thus, the degree of dissociation and solvation of thealkaline earth metal salt within the organic solvent and the cationitself (calcium, magnesium or others) as well as the counterion(chloride, bromide, acetate, etc.) have an influence on the extent ofstabilization: e.g. CaBr₂ shows less effective stabilization compared toCaCI2, caused by the higher atomic radius of bromide compared tochloride. The stabilizing effect of chloride itself without an alkalineearth metal cation (e.g. choline chloride:(2-hydroxyethyl)-trimethylammonium) is shown to be negligible.

More preferably the degradable pharmaceutically active ingredient is analkylating agent, such as a pharmaceutically active ingredient which iscapable of transferring alkyl groups to DNA.

Even more preferred, the degradable active pharmaceutical ingredientcomprises at least one group according to formula (I)

wherein in formula (I)

-   X is Cl or Br, preferably Cl-   R₁ is H, —CH₂CH₂X or —NO, preferably H or —CH₂CH₂X.

Wherein in formula (I) the central nitrogen atom is not substituted by amethyl group, rather the corresponding bond represents the bond to therest of the overall molecule of the degradable active pharmaceuticalingredient.

Particularly preferred, the degradable active pharmaceutical ingredientis a compound according to formula (II)

wherein

-   G is Ar or HetAr-   Ar or HetAr is optionally substituted with 1 to 5, preferably 1 to    4, more preferably 1 to 3, particularly preferred 1 to 2, most    preferably 1 substituent.

The term “Ar” refers to an aromatic cyclic hydrocarbon. Preferably, thearyl group contains 5 to 7 carbon atoms which can be arranged in onering (e.g., phenyl) or two or more condensed rings (e.g., naphthyl).Exemplary aryl groups include cyclopropenylium, cyclopentadienyl,phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, andphenanthryl. Preferably, “aryl” refers to a monocyclic ring containing 6carbon. A preferred example is phenyl.

The term “HetAr” means an aryl group as defined above in which one ormore carbon atoms in the aryl group are replaced by heteroatoms of O, S,or N. Preferably, heteroaryl refers to a five or six-membered aromaticmonocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the sameor different heteroatoms of O, N, or S. Alternatively, it means anaromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5carbon atoms are replaced with the same or different heteroatoms of O,N, or S. Preferably, in each ring of the heteroaryl group the maximumnumber of O atoms is 1, the maximum number of S atoms is 1, and themaximum total number of O and S atoms is 2. Exemplary heteroaryl groupsinclude furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl (1,2,5- and1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-),tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-),pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1,2,3-, 1,2,4-, and 1,3,5-),benzofuranyl (1- and 2-), indolyl, isoindolyl, benzothienyl (1- and 2-),1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl,benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl,isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl(1,2,3- and 1,2,4-benzotriazinyl), pyridazinyl, phenoxazinyl,thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl,chromenyl, xanthenyl, phenoxathiinyl, pyrrolizinyl, indolizinyl,indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl (1,5-,1,6-, 1,7-, 1,8-, and 2,6-), cinnolinyl, pteridinyl, carbazolyl,phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (1,7-, 1,8-,1,10-, 3,8-, and 4,7-), phenazinyl, oxazolopyridinyl,isoxazolopyridinyl, pyrrolooxazolyl, and pyrrolopyrrolyl. Exemplary 5-or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl,isoxazolyl, oxadiazolyl (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl,pyrazolyl, triazolyl (1,2,3- and 1,2,4-), thiazolyl, isothiazolyl,thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyrazinyl,triazinyl (1,2,3-, 1,2,4-, and 1,3,5-), and pyridazinyl.

Most preferably, the at least one easily degradable activepharmaceutical ingredient is selected from the group consisting ofUramustine, Bendamustine, Melphalan, Melflufen, Chlorambucil, preferablyMelphalan, Melflufen, Chlorambucil, Bendamustine, more preferablyBendamustine.

Preferably, the concentration of the at least one degradable activepharmaceutical ingredient in the formulation is 1 to 200 mg/g, morepreferably 5 to 150 mg/g, even more preferably 10 to 100 mg/g, mostpreferably 20 to 40 mg/g based on the overall weight of the formulation.

The at least one pharmaceutically acceptable organic solvent may be anyorganic solvent which is suitable for physiological use.

Preferably the at least one organic solvent is a pharmaceuticallyacceptable polyol. More preferably the at least one pharmaceuticallyacceptable organic solvent is propylene glycol, 1,3-butanediol,polyethylene glycol, such as PEG 400 and/or PEG 300, preferably PEG 400and/or propylene glycol.

In a further embodiment, the at least one pharmaceutically acceptableorganic solvent is a mixture of 5 to 20% Propyleneglycol and 80 to 95%polyethylene glycol (w/w), preferably 10% Propyleneglycol and 90% PEG400 (w/w).

The at least one pharmaceutically acceptable alkaline earth metal saltmay be any pharmaceutically acceptable alkaline earth metal known to theperson skilled in the art. In the context of the present invention,“alkaline earth metal salt” means any ionic compound, includingcoordination complexes and chelate complexes of an alkaline earth metal.

Preferably the at least one earth metal salt is a calcium or a magnesiumsalt. More preferably a calcium salt. Preferably the anion of the atleast one alkaline earth metal salt is gluconate, chloride, bromide,acetate, orotate or lactate more preferably chloride, acetate orlactate, most preferably chloride.

Most preferably the at least one pharmaceutically acceptable alkalineearth metal salt is calcium chloride or magnesium chloride, particularlypreferred calcium chloride.

The at least one pharmaceutically acceptable alkaline earth metal saltis preferably present in a concentration of 0.1 to 3 mol/L, morepreferably 0.1 to 1.5 mol/L, even more preferred 0.3 to 1.5 mol/L,particular preferred 0.7 to 1.1 mol/L, more particular preferred 0.1 to1.0 mol/L, most particular preferred 0.8 to 1.0 mol/L, especiallypreferred 0.1 to 0.5 mol/L based on the overall volume of the liquidformulation.

The at least one pharmaceutically acceptable alkaline earth metal saltin the said concentrations is preferably completely dissolved in thepharmaceutically acceptable organic solvent resulting in a clearpharmaceutical solution.

The liquid pharmaceutical formulation may comprise further additionalingredients known to the person skilled in the art. These may comprisefor example buffers, detergents, preservatives and others.

The liquid pharmaceutical formulation may be applied without furtherdilution or with prior dilution. The pharmaceutical formulation may bediluted with water for injection purposes, saline or buffer solutionbefore administration. Preferably, the pharmaceutical formulation isdiluted with water for injection purposes, saline or buffer solution,not longer than 8 hours, preferably not longer than 4 hours and mostpreferably not longer than 2 hour before administration.

The liquid pharmaceutical formulation may be administered in anysuitable manner known to the person skilled in the art. Preferably, theliquid pharmaceutical formulation is administered parenterally, morepreferably as injection, most preferably as intravenous infusion orintravenous bolus injection.

In one embodiment, the liquid pharmaceutical formulation, preferablybeing essentially free of water comprises

-   a) 1 to 200 mg/g, preferably 5 to 150 mg/g, more preferably 10 to    100 mg/g, most preferably 20 to 40 mg/g based on the overall weight    of the formulation. of the at least one easily degradable active    pharmaceutical ingredient,-   b) at least one pharmaceutically acceptable organic solvent and-   c) the at least one pharmaceutically acceptable alkaline earth metal    salt is preferably present in a concentration of 0.1 to 3 mol/L,    more preferably 0.1 to 1.5 mol/L, even more preferred 0.3 to 1.5    mol/L, particular preferred 0.7 to 1.1 mol/L, more particular    preferred 0.1 to 1.0 mol/L, most particular preferred 0.8 to 1.0    mol/L, especially preferred 0.1 to 0.5 mol/L based on the overall    volume of the liquid formulation based on the overall volume of the    liquid formulation at least one pharmaceutically acceptable alkaline    earth metal salt.

In another embodiment the liquid pharmaceutical formulation, preferablybeing essentially free of water comprises

-   a) at least one easily degradable active pharmaceutical ingredient    of the group consisting of Bendamustine, Chlorambucil, Melphalan,    Melflufen,, preferably Bendamustine,-   b) propylene glycol, 1,3-butanediol, polyethylene glycol, such as    PEG 400 and/or PEG 300, preferably PEG 400 and/or propylene glycol.-   c) at least calcium chloride or magnesium chloride, preferably    calcium chloride.

In a further embodiment the liquid pharmaceutical formulation,preferably being essentially free of water comprises

-   a) 1 to 200 mg/g, preferably 5 to 150 mg/g, more preferably 10 to    100 mg/g, most preferably 20 to 40 mg/g based on the overall weight    of the formulation. of at least one easily degradable active    pharmaceutical ingredient of the group consisting of Bendamustine,    Chlorambucil, Melphalan, Melflufen,, preferably Bendamustine,-   b) propylene glycol, 1,3-butanediol, polyethylene glycol, such as    PEG 400 and/or PEG 300, preferably PEG 400 and/or propylene glycol    and-   c) 0.1 to 3 mol/L, more preferably 0.1 to 1.5 mol/L, even more    preferred 0.3 to 1.5 mol/L, particular preferred 0.7 to 1.1 mol/L,    more particular preferred 0.1 to 1.0 mol/L, most particular    preferred 0.8 to 1.0 mol/L, especially preferred 0.1 to 0.5 mol/L    based on the overall volume of the liquid formulation of at least    calcium chloride or magnesium chloride, preferably calcium chloride.

In another embodiment the liquid pharmaceutical formulation, preferablybeing essentially free of water comprises

-   a) at least one active pharmaceutical ingredient of the group    consisting of Bendamustine, Melphalan, Melflufen, Chlorambucil, and    Uramustine.-   b) polyethylene glycol, such as PEG 400 and/or PEG 300, propylene    glycol, 1,3-butanediol, preferably polyethylene glycol, propylene    glycol or mixtures thereof and-   c) at least calcium chloride or magnesium chloride, preferably    calcium chloride.

In a further embodiment the liquid pharmaceutical formulation,preferably being essentially free of water comprises

-   a) 1 to 200 mg/g, preferably 5 to 150 mg/g, more preferably 10 to    100 mg/g, most preferably 20 to 40 mg/g based on the overall weight    of the formulation. of at least one active pharmaceutical ingredient    of the group consisting of Bendamustine, Melphalan, Melflufen,    Chlorambucil, and Uramustine, preferably Bendamustine,-   b) propylene glycol, 1,3-butanediol, polyethylene glycol, such as    PEG 400 and/or PEG 300, preferably PEG 400 and/or propylene glycol    and-   c) 0.1 to 3 mol/L, more preferably 0.1 to 1.5 mol/L, even more    preferred 0.3 to 1.5 mol/L, particular preferred 0.7 to 1.1 mol/L,    more particular preferred 0.1 to 1.0 mol/L, most particular    preferred 0.8 to 1.0 mol/L, especially preferred 0.1 to 0.5 mol/L    based on the overall volume of the liquid formulation of at least    calcium chloride or magnesium chloride, preferably calcium chloride.

In a further embodiment the liquid pharmaceutical formulation,preferably being essentially free of water comprises

-   a) at least one easily degradable active pharmaceutical ingredient,    wherein the degradable active pharmaceutical ingredient is a    compound according to formula (II)

-   

-   wherein    -   G is Ar or HetAr    -   Ar or HetAr is optionally substituted with 1 to 5, preferably 1        to 4, more preferably 1 to 3, particularly preferred 1 to 2,        most preferably 1 substituent,

-   b) propylene glycol, 1,3-butanediol, polyethylene glycol, such as    PEG 400 and/or PEG 300, preferably PEG 400 and/or propylene glycol    and

-   c) at least calcium chloride or magnesium chloride, preferably    calcium chloride.

Moreover the present invention is directed to the use of the liquidpharmaceutical formulation as defined above, for use in medicine.

The inventive liquid pharmaceutical formulation may be combined with aphysiologically acceptable aqueous solution prior to application to thepatient. For example the liquid pharmaceutical formulation may becombined with suitable amount of isotonic sodium chloride solution.Preferably the concentration of the isotonic sodium chloride solutioncomprises 9 mg/L sodium chloride and preferably exhibits a pH value of4.5 to 7.0.

The present invention further comprises a formulation comprising a) theinventive liquid pharmaceutical formulation, as described above and b) aphysiologically acceptable aqueous solution, as well as its use inmedicine.

Furthermore, the present invention is directed to the use of the liquidpharmaceutical formulation as defined above, optionally in the presenceof water, for use in the treatment of cancer.

Use of at least one pharmaceutically acceptable alkaline earth metalsalt in a liquid pharmaceutical formulation, preferably beingessentially free of water comprising

-   a) at least one easily degradable active pharmaceutical ingredient,-   b) at least one pharmaceutically acceptable organic solvent and-   c) the at least one pharmaceutically acceptable alkaline earth metal    salt, for the stabilization of the at least one easily degradable    active pharmaceutical ingredient.

The term “stabilization” in the context of the invention meansprevention or reduction of degradation of the easily degradable activepharmaceutical ingredient. Preferably it means maintaining a purity asdetermined by suitable analytical methods of at least 85%, morepreferably of at least 90%, most preferably of at least 95% at the endof the defined shelf life of pharmaceutical preparation, in particularcompared with a not stabilized sample. For example, the purity may bedetermined by high-performance-liquid-chromatography (HPLC), inparticular reversed phase high-performance liquid-chromatography(RP-HPLC). Furthermore stabilization means that any visible color changeof the preparation caused by degradation is prevented or reduced duringproduct shelf life, in particular in comparison with a not stabilizedsample.

EXAMPLES OF THE INVENTION

The invention is further illustrated in the following Examples.

Example 1: Determination of Bendamustine Quantity and Purity by ReversedPhase Chromatography (RP-HPLC)

The purity of Bendamustine hydrochloride and quantity was determined asfollows using a standard curve:

Column type: C18, 100 × 4.6 mm, 2.6 µm Mobile phase: A: Water/ACN/TFA =950/50/1 v/v/v B: Water/ACN/TFA = 50/950/1 v/v/v Column temperature: 25°C. Autosampler temperature: 25° C. Flow rate: 1.2 mL/min Detectionwavelength: 234 nm

HPLC Gradient

TABLE 1 HPLC Gradient Time Eluent A Eluent B [min] 0 95% 5% 5.00 95% 5%9.00 80% 20% 25.00 55% 45% 28.00 20% 80% 30.00 20% 80% 30.01 95% 5%35.00 95% 5%

Example 2: Stabilizer Solution Study

The solubility of potential stabilizers to be tested forBendamustine-HCI liquid formulation was evaluated in polypropyleneglycol. The respective chemical was dissolved in water under continuousstirring at 40° C. After complete dissolution the solutions were storedat 2° C. - 5° C. over-night before analysis to simulate the worst case.

TABLE 2 Stabilizer solubility study results Stabilizer Targetconcentration [mol/kg] Outcome CaCl₂ 0.9 Complete dissolution CaBr₂ 0.9Complete dissolution MgCl₂ 0.9 Complete dissolution Choline-HCI 1.8Complete dissolution

Example 3: Bendamustine HCl Solubility Study

The solvent systems w/ and w/o potential stabilizers listed in Table 3were compounded. Bendamustine-HCI drug substance was added. The sampleswere stirred at room temperature and stored overnight at 2-8° C. Theconcentration of Bendamustine-HCI in each system was quantified aftercentrifugation in the supernatant by RP-HPLC (Example 1).

TABLE 3 Formulation variants tested for Bendamustine-HCl solubilityFormulation variant Sample description Comment Benda HCl [g] Solvent [g]Max. Solubility at 2-8° C. [mg/g] 1 Mix, according to WO 2010/036702A1:, 90% PEG 400 / 10% PG (w/w) benchmark 0.80 9.20 69 2 PG solventnegative control 0.80 9.20 69 3 PG, molecular sieve, filtrated dryingagent 0.80 9.20 74 4 PG, molecular sieve, permanent contact drying agent0.80 9.20 72 5 PG + CaCl₂ stabilizer 0.80 9.20 55 6 PG + CaBr₂stabilizer 0.80 9.20 17 7 PG + Cholin/HCl Cl⁻ source, according toUS9,572,797 0.80 9.20 70 8 PG + Cholin (70 mM) Patent control 0.80 9.2080

The target concentration for Bendamustine-HCI was 80 mg/g. The detectedconcentrations in the specific formulations after storage over-night at2-8° C. and subsequent centrifugation are determined by means of RP-HPLCusing a standard curve.

Example 4: Bendamustine HCl Stability Study 1

Bendamustin containing compositions were prepared by dissolvingBendamustine-HCI to a concentration of 25 mg/gin one of the formulations#1 to #10 as listed below in Table 4.

Samples were stored at 25° C. and 40° C. for 60 days. Content and puritywas determined at T=0 and at further time points.

TABLE 4 Formulation variants tested for Bendamustine-HCl stabilityFormulation variant Sample description comment purity after 60 d at 25°C. purity after 60 d at 40° C. 1 Mix, according to WO 2010/036702 A1:,90% PEG 400 / 10% PG (w/w) benchmark 94.1% 85.9% 2 PG solvent negativecontrol 4.3% 1.1% 3 PG, molecular sieve, filtrated drying agent 94.3%1.1% 4 PG, molecular sieve, permanent contact drying agent 84.5% 59.1% 5PG + 0.9 mol/kg CaCl₂ drying agent 98.6% 94.7% 6 PG + 0.9 mol/kg CaBr₂drying agent 73.6% 25.2% 7 PG + 1.8 mol/kg Cholin-HCI Cl- source 6.5%1.4% 8 PG + 70 mmol/kg Cholin-HCI according to US 9,572,797 6.0% 1.6% 9PG + 0.9 mol/kg MgCl₂ drying agent 97.4% 89.7% 10 Mix, according to:WO2011/094565 w/ thiogylcerol, 90% PEG 400 / 10% PG /0.5%monothioglycerol (w/w) Benchmark, market product 97.7% 88.1%

Purity of Bendamustine-HCI was assessed by evaluating relative main peakareas in RP-HPLC chromatograms. For detailed results see FIG. 2 to FIG.5 .

Formulation variant 5 (PG + 0.9 mol/kg CaCl2) showed highest stabilityof Bendamustine-HCI after 60 days of storage at 25° C. as well as at 40°C., respectively. Specifically, the purity in this formulation variant 5at the end of the study was determined as 98.6% at 25° C. and 94.7% at40° C. Formulations 1, 9, and 10 also showed respectable results with85.9% (variant 1), 89.7% (variant 9), and 88.1% (variant 10) purity at40° C. Variant 3 (PG + molecular sieve filtered) showed good results at25° C. with 94.3% purity at the end of the study but then rather badresults at 40° C. with nearly complete degradation during storage (1.1%main peak area). The purity values determined for variant 4 (PG +molecular sieve) showed high fluctuations and were inconsistent whichmay be due to the rather undefined composition of the formulationcontaining “a few” sieve pellets per vial.

Example 5: Visual Appearance of Bendamustine HCl Preparation

Due to changes of visual appearance of the samples further underling theRP-HPLC results, macro photographs of vials were taken after the end ofthe stability study. FIG. 6 shows two photographs comparing allvariants.

Again, the superiority of formulation variant 5 (PG + 0.9 mol/kg CaCl2)over all other variants is apparent as no color change at all could bedetected. All other variants show at least a slight yellow tint(formulation variants 6, 9, and 10) which may be interpreted as somekind of degradation. Interestingly, formulation variants 1 and 10(benchmark formulation w/ and w/o monothioglycerol) also show a ratherstrong yellow tint, whereby it seems more pronounced in the formulationwithout monothioglycerol (formulation variant 1).

Example 6: Bendamustine HCl Stability Study 2: Comparative Examples

The following Bendamustine containing compositions (25 mgBendamustine/HCI in 4 g solution) were prepared, filled into type 1glass vials. The closed vials were stored at defined temperatures (2-8°C., 25° C. and 40° C.) as shown in the table below.

At defined time point (4 weeks) samples were taken and analyzed be mansof the RP-HPLC method described in Example 1.

TABLE 5 Stability data for 2-8° C., 25° C. and 40° C. Solventcomposition Purity after T=4 weeks [%]¹ 2-8° C. 25° C. 40° C. ZnCI2 0.9mol/kg in PG 96.5 40.9 3.3 ZnCl₂ 0.45 mol/kg in PG 96.4 41.3 3.2 KAc 0.9mol/kg in PG 59.4 0.9 0.3 ZnCl₂ 0.224 mol/kg in PEG400/PG 90/10 99.5²96.4² 84.4² ¹Bendamustin API: 99.5% purity ²Purity after T = 3 weeks [%]

Conclusion: The salts ZnCl₂ and KAc, both not belonging to the class ofalkaline earth metal salts, are soluble in organic solvents but show nosignificant stabilizing effect on Bendamustine neither in the propyleneglycol nor in the PEG400/PG solvent mixture.

Compared to the degradation rate of Bendamustine in PEG400/PG withoutaddition of mineral salt (see example 8, the degradation of Bendamustinein the PEG400/PG solvent mixture is even more pronounced in presence ofco-dissolved ZnCl₂.

Example 7: Stability Study of Other Active Pharmaceutical Ingredients

In example 2 it was shown that calcium chloride in propylene glycol hada stabilizing effect on Bendamustine-HCl. To further investigate thisphenomenon, other pharmaceutically active molecules with differentdegradation mechanisms were tested for stability in a 4 weeks short termstability experiment at 40° C. To serve as a direct reference,Bendamustine-HCI was also included in the study. To differentiatebetween the stabilizing effects, if any, between calcium and chlorideall experiments were run in parallel with addition of choline-chloridein the same molar amount in respect to chloride to demonstrate that thestabilizing effect - if any - is attributed to calcium not chloride.

The following compositions were prepared, filled into type 1 glassvials, closed with a rubber stopper and crimp caped. The closed vialswere stored at a temperature of 40° C.

TABLE 6 Substances tested in a short term stability study at 40° C. APIconc. [mg/g] formulation comment Bendamustine 25 CaCl₂ 99.9 mg | PG ad1000 mg to confirm previous results Bendamustine 25 Choline/HCl 251.3 mg| PG ad 1000 mg Cl-source Carmustine 35 CaCl₂ 99.9 mg I PG ad 1000 mgAPI with untypical N-mustard structure (β-chloro-nitrosourea) Carmustine35 Choline/HCl 251.3 mg | PG ad 1000 mg Cl-source Azacytidine 25 CaCl₂99.9 mg | PG ad 1000 mg API w/o N-mustard but sensitive to hydrolysisAzacytidine 25 Choline/HCl 251.3 mg | PG ad 1000 mg Cl-source Melphalan5.0 CaCl₂ 99.9 mg | PG ad 1000 mg API with arom. N-mustard structureMelphalan 5.0 Choline/HCl 251.3 mg | PG ad 1000 mg Cl-source

At defined time points samples were taken and analyzed be mans of theRP-HPLC method described in Example 1.

The following results for content (concentration of API) and purity ofthe APIs were obtained:

Bendamustine

The results generated in example 2 could be confirmed. After 4 weeks ofstorage Bendamustine showed a purity of roughly 95% in the calciumchloride formulation and only about 2% in the Choline-HCI formulation.The concentration dropped accordingly. See FIGS. 7 and 8 , as well astables 7 and 8.

TABLE 7 Purity of Bendamustine at 40° C. as relative main peak area [%]by RP-HPLC Formulation variant # Time [days] CaCl₂ Choline HCl 0 99.7%99.6% 7 98.9% 78.2% 14 98.4% 26.1% 28 95.3% 1.8%

TABLE 8 Rel. content of Bendamustine at 40° C. determined by RP-HPLCusing a calibration curve Formulation variant Time [days] CaCl₂ CholineHCl 0 100% 100% 7 98.9% 78.5% 14 98.4% 24.4% 28 95.3% 1.3%

Azacitidine

Azacitidine degraded rapidly in both formulations. Calcium chlorideshowed no stabilizing effect on Azacytidine, as shown in Tables 9, 10and FIGS. 9, 10 .

TABLE 9 Purity of Azacitidine at 40° C. as relative main peak area [%]by RP-HPLC Formulation variant Time [days] CaCl₂ Choline HCl 0 100% 100%7 99.8% 99.8% 14 97.7% 99.5% 28 0.0% 0.0%

Carmustine

Carmustine was completely degraded already after 1 week in bothformulations. Calcium chloride showed no stabilizing effect onCarmustine. See FIGS. 11, 12 and tables 10, 11.

TABLE 10 Purity of Carmustine at 40° C. as relative main peak area [%]by RP-HPLC Formulation variant Time [days] CaCl₂ Choline HCl 0 95.8%96.1% 7 2.1% 1.6% 14 2.0% 2.4% 28 1.9% 2.2%

TABLE 11 Rel. content of Carmustine at 40° C. determined by RP-HPLCusing a calibration curve Formulation variant Time [days] CaCl₂ CholineHCl 0 100% 100% 7 3.6% 2.6% 14 1.3% 1.4% 28 1.3% 1.2%

Melphalan

Melphalan showed a relatively high stability in the solvent even withoutstabilizer. However, content and purity of the preparation with calciumchloride was higher than with choline chloride. See FIGS. 13, 14 andtables 12, 13.

TABLE 12 Purity of Melphalan at 40° C. as relative main peak area[%] byRP-HPLC Formulation variant Time [days] CaCl₂ Choline HCl 0 99.8% 96.7%7 98.1% 96.6% 14 97.4% 92.4% 28 95.4% 91.0%

TABLE 13 Rel. content of Melphalan at 40° C. determined by RP-HPLC usinga calibration curve Formulation variant Time [days] CaCl₂ Choline HCl 0100% 100% 7 84.6% 86.1% 14 86.2% 74.0% 28 86.5% 76.9%

Conclusions

The stabilizing effect of calcium-chloride on the typical N-mustardcompound Bendamustine could be reproduced. The stabilizing effect can beclearly attributed to calcium and is not conditioned by chloride aschloride (given as Choline-chloride) has no stabilizing effect (completedegradation within 2 to 4 weeks).

Similar results were seen with Melphalan but due to the per se higherstability of Melphalan compared to Bendamustine the results are lessdifferentiating.

Azacitidine, which is not a N-mustard substance is not stabilized bycalcium.

Carmustine, which is not an aromatic nitrogen mustard but aβ-chloro-nitrosourea is also not stabilized by calcium.

Example 8: Dose Dependent Stabilization Effect of Calcium ChlorideDissolved in PEG400

The objective of this study was to evaluate the dose depending efficacyof calcium chloride to reduce degradation products during stress storageof Bendamustine-HCI organic liquid formulations. The organic solventsystems PEG400 with a small amount of alcoholic solvents 1,2-propanediolor ethanol (10% w/w) are to be investigated.

The following Bendamustine containing compositions were prepared. Allpreparations were clearly and colorless soluble and could be sterilefiltered through a 0.22 µm PES sterile filter. The preparations werefilled into type 1 glass vials and stoppered with nitrogen overlay. Theclosed vials were stored at 40° C.:

TABLE 14 # Bendamusti ne-HCl Polyethylen e glycol 400 1,2-PropanediolEthanol CaCl₂ 1-Thioglycerol 1 0.10 g 3.44 g 0.40 g - 0.056 g - 2 0.10 g3.40 g 0.40 g - 0.099 g - 3 0.10 g 3.36 g 0.40 g - 0.141 g - 4 0.10 g3.64 g - 0.20 g 0.056 g - 5 0.10 g 3.60 g - 0.20 g 0.099 g - 6 0.10 g3.50 g 0.40 g - - - 7 0.10 g 3.70 g - 0.20 g - - 8 0.10 g 3.50 g 0.40g - - 0.005 g

At defined time point (4 weeks) samples were taken and analyzed be mansof the RP-HPLC method described in Example 1.

The following results were obtained:

Purity by RP-HPLC

The purity of Bendamustine-HCI was assessed via RP-HPLC and expressed byrelative main peak area. Measurements were carried out in duplicate.

TABLE 15 Purity of Bendamustine-HCl at 40° C. as relative main peak area[%] by RP-HPLC Formulation variant # Time [weeks ] Meas . # 1 2 3 4 5 67 8 0 1 99.8 99.8 99.8 99.8 99.8 99.8 99.7 99.9 2 99.8 99.8 99.8 99.799.8 99.9 99.7 99.9 4 1 95.3 96.2 96.6 94.7 95.2 93.3 91.7 93.5 2 95.396.1 96.6 94.5 95.4 93.2 91.6 93.5 8 1 94.6 95.4 95.7 93.7 94.1 92.390.8 91.9 2 94.6 n.a. 95.8 93.7 94.2 92.2 91.0 91.9

As displayed in the graph of FIG. 15 , the purity was calculatedrelatively to the value obtained at T=0 (i.e. T=0 ₌ 100%).

Content by RP-HPLC

The content of Bendamustine-HCI was assessed via RP-HPLC using a 25 mg/gBendamustine-HCI standard as reference. Standard solutions were freshlyprepared for each individual sampling time point. Standard curves wererecorded in duplicate at the beginning and at the end of each sequence.Samples were analyzed in duplicate.

TABLE 16 Content [mg/g] of Bendamustine-HCl at 40° C. by RP-HPLCFormulation variant # Time [weeks ] Meas . # 1 2 3 4 5 6 7 8 0 1 25.324.9 25.2 25.6 25.2 24.8 25.1 24.7 2 25.6 25.4 25.2 25.6 24.8 25.0 25.124.7 4 1 23.4 23.7 24.1 23.3 23.5 22.1 21.8 22.6 2 23.3 23.5 24.2 23.223.4 22.4 22.0 22.6 8 1 22.3 23.0 23.2 22.2 22.9 21.7 21.2 21.7 2 22.9n.a. 23.2 22.4 22.9 21.8 21.1 21.8 Target: 25 mg/g

In the graph shown in FIG. 16 the content after 8 weeks was calculatedrelatively to the value obtained at T=0 (i.e. T=0 ₌ 100%).

In FIGS. 17 and 18 , graphs relating purity and content of Bendamustineare shown over storage time (storage temperature 40° C.). Shown are datafor the more relevant solvent system PEG 400 / PG.

Visual Appearance

The visual appearance of the liquid formulations was documented by macrophotography. Photographs of representative vials after 8 weeks storagetime at 40° C. are displayed (FIG. 19 ).

Discussion

A clear dose depending stabilizing effect of calcium chloride onBendamustine could be shown for both solvent mixtures. Content, purityand colorlessness of Bendamustine preparations are better maintainedwhen calcium chloride is added. The solvent system PEG400/PG is superiorto PEG400/ethanol, which showed higher degradation rates and coloration.The stabilizer calcium chloride proved to be superior to thioglycerol,which showed a negligible effect only.

Moreover, the invention is directed to the following items:

1. A liquid pharmaceutical formulation, being essentially free of water,comprising

-   a) at least one easily degradable active pharmaceutical ingredient,-   b) at least one pharmaceutically acceptable organic solvent and-   c) at least one pharmaceutically acceptable alkaline earth metal    salt.

2. The liquid pharmaceutical formulation according to item 1, whereinall components of the formulation are completely dissolved resulting ina clear solution.

3. The liquid pharmaceutical formulation according to item 1 or 2,wherein the at least one alkaline earth metal salt is a calcium salt.

4. The liquid pharmaceutical formulation according to any one of items 1to 3, wherein the at least one alkaline earth metal salt is a magnesiumsalt.

5. The liquid pharmaceutical formulation according to any one of items 1to 4, wherein the anion of the at least one alkaline earth metal salt isgluconate, orotate, chloride, bromide, acetate or lactate, preferablychloride.

6. The liquid pharmaceutical formulation according to any one of theitems 1 to 5, wherein the at least one pharmaceutically acceptableorganic solvent is propylene glycol, 1,3-butanediol, polyethylene glycolsuch as PEG 400 and/or PEG 300, preferably propylene glycol and/or1,3-butanediol.

7. The liquid pharmaceutical formulation according to any one of items 1to 6, wherein the at least one pharmaceutically acceptable alkalineearth metal salt is present in a concentration of 0.1 to 3 mol/L,preferably 0.3 to 1.5 mol/L, more preferably 0.7 to 1.1 mol/L.

8. The liquid pharmaceutical formulation according to any one of items 1to 7, wherein the easily degradable active pharmaceutical ingredient issensitive to hydrolysis.

9. The liquid pharmaceutical formulation according to any one of theitems 1 to 8, wherein the easily degradable active pharmaceuticalingredient is an alkylating agent.

10. The liquid pharmaceutical formulation according to any one of items1 to 9, wherein the easily degradable active pharmaceutical ingredientcomprises at least one group according to formula (I)

wherein in formula (I),

-   X is Cl or Br, preferably Cl-   R₁ is H, —CH₂CH₂X or —NO, preferably H or —CH₂CH₂X.

11. The liquid pharmaceutical formulation according to any one of items1 to 10, wherein the at least one easily degradable activepharmaceutical ingredient is present in a concentration of 1 to 200mg/g, preferably 5 to 150 mg/g, more preferably 10 to 100 mg/g, mostpreferably 20 to 40 mg/g based on the overall weight of the formulation.

12. The liquid pharmaceutical formulation according to any one of items1 to 11, wherein the at least one easily degradable activepharmaceutical ingredient is selected from the group consisting ofCarmustine, Lomustine, Nimustine, Bendamustine, Cyclophosphamide,Melphalan, Melflufen, Azacytidine, Chlorambucil, Ifosfamide,Procarbazine, Dacarbazine, Tremozolomide, Mechlorethamine, Thiotepa,Cisplatin, Carboplatin, Oxaliplatin, Busulfan, Treosulfan, andFosaprepitant, preferably consisting of Bendamustine, Chlorambucil,Carmustine, Lomustin, Cyclophosphamide, Melphalan, Melflufen,Ifosfamide, Mechlorethamine, more preferably consisting of Bendamustine.

13. The liquid pharmaceutical formulation, according to any one of items1 to 12, optionally in the presence of water, for use in medicine.

14. The liquid pharmaceutical formulation, according to any one of items1 to 13, optionally in the presence of water, for use in the treatmentof cancer.

15. Use of at least one pharmaceutically acceptable alkaline earth metalsalt in a liquid pharmaceutical formulation, being essentially free ofwater, comprising

-   a) at least one easily degradable active pharmaceutical ingredient,-   b) at least one pharmaceutically acceptable organic solvent and-   c) the at least one pharmaceutically acceptable alkaline earth metal    salt.

for the stabilization of the at least one easily degradable activepharmaceutical ingredient.

The invention illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by exemplary embodiments and optional features, modificationand variation of the inventions embodied herein may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed is:
 1. A method of treating a subject having cancer, themethod comprising administering to the patient a therapeuticallyeffective amount of a liquid pharmaceutical formulation, having a watercontent of below 1.0% (w/w) water, comprising a) Bendamustine as anactive pharmaceutical ingredient; b) at least one pharmaceuticallyacceptable organic solvent comprising propylene glycol, polyethyleneglycol 400 (PEG 400) or a mixture of propylene glycol and PEG 400; andc) a calcium salt.
 2. The method of claim 1, wherein the liquidpharmaceutical formulation is administered parenterally.
 3. The methodof claim 2, wherein the liquid pharmaceutical formulation isadministered as intravenous infusion or intravenous bolus injection. 4.The method of claim 1 wherein the liquid pharmaceutical formulation isdiluted prior to administration.
 5. The method of claim 4, wherein theliquid pharmaceutical formulation is diluted with water for injection,saline or a buffer solution prior to administration.
 6. The method ofclaim 5, wherein the liquid pharmaceutical formulation is diluted withwater for injection, saline or a buffer solution not longer than 8hours, no longer than 4 hours or not longer than 2 hours prior toadministration.
 7. The method of claim 1, wherein all components of theliquid pharmaceutical formulation are completely dissolved resulting ina clear solution.
 8. The method of claim 1, wherein the anion of thecalcium salt in the liquid pharmaceutical formulation is gluconate,chloride, bromide, acetate or lactate.
 9. The method of claim 1, whereinthe at least one pharmaceutically acceptable organic solvent of theliquid pharmaceutical formulation is a mixture of propylene glycol andPEG
 400. 10. The method of claim 1, wherein the at least onepharmaceutically acceptable organic solvent of the liquid pharmaceuticalformulation comprises at least 80% PEG
 400. 11. The method of clam 1,wherein the calcium salt is present in a concentration of 0.1 to 3 mol/Lbased on the overall volume of the liquid pharmaceutical formulation.12. The method of claim 1, wherein the Bendamustine is present in aconcentration of 1 to 200 mg/g based on the overall weight of theformulation.
 13. The method of claim 12, wherein the at least onepharmaceutically acceptable organic solvent of the liquid pharmaceuticalformulation is a mixture of 5 to 20% propylene glycol and 80 to 95% PEG400.
 14. The method of claim 13, wherein the at least onepharmaceutically acceptable organic solvent of the liquid pharmaceuticalformulation is a mixture of 10% propylene glycol and 90% PEG 400 (w/w).15. The method of claim 11, wherein the calcium salt is a chloride. 16.The method of claim 1, wherein the calcium salt is present in aconcentration of 0.1 to 1.5 mol/L based on the overall volume of theliquid pharmaceutical formulation.
 17. The method of claim 1, whereinthe calcium salt is present in a concentration of 0.3 to 1.5 mol/L,based on the overall volume of the liquid pharmaceutical formulation.18. The method of claim 1, wherein the calcium salt is present in aconcentration of 0.1 to 0.5 mol/L based on the overall volume of theliquid pharmaceutical formulation.
 19. The method of claim 1, whereinthe Bendamustine is present in a concentration of 5 to 150 mg/g based onthe overall weight of the liquid pharmaceutical formulation.
 20. Themethod of claim 1, wherein the Bendamustine is present in aconcentration of 10 to 100 mg/g based on the overall weight of theliquid pharmaceutical formulation.
 21. The method of claim 1, whereinBendamustine is present in a concentration of 20 to 40 mg/g based on theoverall weight of the liquid pharmaceutical formulation.
 22. The methodof claim 1, wherein the patient is a human.